Immunocytochemical staining and cell immunoblot assay (CIBA) were performed in adenoma tissue from five patients with Cushing's disease and three patients with clinically silent corticotroph adenomas. All five patients with Cushing's disease showed hypersecretion of ACTH (130, 190, 331, 120, and 130 pg/ml), high levels of serum cortisol (26.6-44.0 μg/dl), and symptoms of Cushing's disease. All three patients with silent corticotroph adenoma showed hypersecretion of ACTH (110, 140, and 160 pg/ml) and normal levels of serum cortisol (11.4-26.8 μg/dl). The size of the pituitary adenoma on magnetic resonance imaging was smaller in patients with Cushing's disease (mean 8.2 mm) than in patients with silent corticotroph adenoma (mean 26.7 mm) (p=0.001). Transsphenoidal surgery was performed to totally resect the adenoma tissue. Immunostaining for ACTH showed diffuse ACTH-immunopositive cells in all eight adenomas. CIBA technique showed a good correlation between percentage of ACTH-immunopositive cells and level of plasma ACTH in patients with Cushing's disease but no correlation between the two parameters in patients with silent corticotroph adenoma. The percentage of ACTH-secreting cells and the amount of hormone secreted by a single cell are too low in silent corticotroph adenomas to cause an increase in plasma ACTH level corresponding to the large tumor size.

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To determine the clinical utility of thyroid ultrasonography in the diagnosis of congenital hypothyroidism (CH) before initiation of therapy, ultrasonographic images of the thyroid gland with a high-resolution transducer were obtained in 204 healthy infants aged from newborn to 12 months (Group A), and 174 infants suspected of having CH detected by neonatal mass screening (Group B). The thyroid gland was imaged by transverse scanning at the anatomic site of the thyroid gland. The maximal width of thyroid on the transverse section in the normal location was measured. By comparing with the normal thyroid gland size and location obtained from Group A, 174 infants of Group B were divided into four subgroups: 1) Normal in size (n=117), 2) Enlarged (n=33), 3) Small (n=1) and 4) Invisible in the normal location (n=23). They were compared with the final diagnoses based on the results of chemical laboratory data and scintigraphic findings. The sensitivity and the specificity for the presence or absence of the thyroid gland in the normal location were 96% (22/23) and 99% (150/151), respectively. Both subgroups of normal and enlarged sized gland included healthy infants (false positive), transient hyperthyrotropinaemia, transient hypothyroidism and CH due to dyshormonogenesis. We conclude that ultrasonography is useful for determining the presence or absence of the thyroid gland in the normal location, whereas normal and enlarged sized glands require further examination to complete the diagnosis.

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Nutrition plays an important role in regulating the circulating levels of insulin-like growth factor-I (IGF-I). We have demonstrated that reduced nitrogen balance is related to changes in serum IGF-I levels but not serum growth hormone levels in pregnant rats. In the present study, we investigated the effects of changes in nitrogen balance on serum IGF-I levels in normal and malnourished pregnant women (defined as having negative nitrogen balance). Forty-eight pregnant women (threatened miscarriage and premature labor, 39; pre-eclampsia, 3; hyper-emesis, 3; ileus, 2; bleeding from the colon, 1) and 19 non-pregnant women admitted in Kobe University Hospital were enrolled in this study. Blood samples of normal pregnant controls were also obtained from 172 healthy pregnant women attending the outpatient clinic. Serum IGF-I levels and nitrogen balance were measured serially in 9 pregnant women with threatened miscarriage and premature labor and 9 malnourished pregnant women out of 48 pregnant women. Serum IGF-I and urinary nitrogen levels were measured by radioimmunoassay with acid-ethanol extraction and the Dumas method, respectively. Nitrogen balance was expressed as the difference between daily nitrogen intake and nitrogen excretion assessed by urinary nitrogen levels. Serum IGF-I levels in normal pregnant controls significantly increased in the third trimester of pregnancy compared with non-pregnant controls. No difference in serum IGF-I levels in any trimester of pregnancy was observed between normal pregnant controls and pregnant women with threatened miscarriage and premature labor. There was no significant difference in nitrogen balance between the pregnant women with threatened miscarriage and premature labor and non-pregnant controls. In the longitudinal study, no correlation was found between the changes in serum IGF-I levels and those in nitrogen balance in the 9 pregnant women with threatened premature labor (daily nitrogen balance > 0 g/day) on the basis of linear regression analysis. On the other hand, the changes in serum IGF-I levels in the 9 malnourished pregnant women were significantly correlated with those in nitrogen balance (y=1.72x+17.5; r=0.60; P<0.05: linear regression analysis). These results indicate that maternal nutritional states have a major effect on serum IGF-I levels in malnourished pregnant women, but not in pregnant women with daily nitrogen balance > 0 g/day. Serum IGF-I levels can be a potent index of nutritional states under malnutrition during human pregnancy.

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This study investigated whether symptoms and findings of hyperthyroidism exist in patients with subclinical hyperthyroidism (SCH) and sought to determine whether hyperthyroidism treatment improves them. Twenty patients (mean age: 36.10±1.41 years) and 20 healthy controls [mean age: 36.35±1.50 years) were included in the study. The SCH duration of patients was at least 6 months. Bone mineral density (BMD) was measured in both patients and controls. The patients were randomly divided into 2 groups of 10 patients each. Symptoms and findings of hyperthyroidism were evaluated and BMD, 24 hour ambulatory blood pressure, holter measurements and serum lipids were determined initially in both groups and 6 months after the attainment of euthyroidism in the treatment group (Group 1) and after a 6 month follow-up in the observation group (Group 2). In the patient group, BMD showed a decrease of 1.3% and 3.9% in femur neck and L_1-4 vertebra compared with controls, respectively. But there was no difference in BMD between patients and controls. Fatigue, nervousness, over sweating, tachycardia and tremor improved with treatment. The number of patients with fatigue, nervousness, over sweating and tachycardia increased in Group 2 after the observation. There was no difference between initial values and after a 6 month period from observation or on attainment of euthyroidism in the values of BMD, lipids, minimal and maximal heart rate, total number of ventricular and supraventricular beats and heart rate variability. As a result symptoms of hyperthyroidism were found to be increased in SCH but they partly decreased after antithyroid treatment. But no favourable effects of antithyroid treatment on BMD, heart rate and arrhythmia incidence were found in young, premenopausal patients with SCH during the 6 month period.

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A 33-year old female was diagnosed as Graves' disease and started on carbimazole. One month later when she was already euthyroid only on carbimazole therapy, she developed acute pancreatitis associated with mild cholestatic hepatitis and erythema nodosum. Carbimazole therapy was interrupted, pancreatic and liver function gradually improved and became normalized two weeks later. Other potential etiological causes of acute pancreatitis, hepatitis and erythema nodosum were excluded. Rechallenge with a single dose of carbimazole led to a new episode of acute pancreatitis and cholestatic hepatitis one day later. The appearance of different hypersensitivity reactions including pancreatitis, hepatitis and erythema nodosum, together with the observation that the interval between drug intake and onset of symptoms became shorter with repeated exposure to carbimazole, point to an immune-mediated mechanism. Carbimazole has to be added to the list of drugs capable of inducing acute pancreatitis, and should be emphasized the need to discontinue this medication as soon as there is evidence of pancreatic dysfunction.

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We report two cases of type 1 diabetes mellitus fulminantly developed as diabetic ketoacidosis (DKA) at 19 weeks of gestation and immediately after delivery. Development of type 1 diabetes around pregnancy is not rare, but its fulminant development is highly uncommon. We also review the relevant literature concerning mostly Japanese cases. In our cases, the group of patients with fulminant progressive diabetes mellitus associated with pregnancy required insulin replacement therapy even after the acute period and showed high value of pancreatic exocrine enzymes, i.e. amylase, elastase, and lipase. The phenotype of this group was similar to “nonautoimmune, fulminant type 1 diabetes” described by Imagawa et al., where the laboratory data of type 1 diabetes-related autoantibodies showed negative. It is well known that autoimmune diseases are in good control during pregnancy. Our present finding suggests that this type of fulminant type 1 diabetes mellitus associated with pregnancy might develop as a consequence of a nonautoimmune mechanism.

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The objective of this study was to clarify the influence of pioglitazone (Pio) on proinsulin (PI) in patients with type 2 diabetes mellitus. The subjects were 55 patients with type 2 diabetes. Among them, 18, 18, and 19 patients were respectively treated with Pio alone (group P), gliclazide (Gli) alone (group G), or Pio plus Gli (group PG) for 12 weeks. Fasting blood samples were obtained before and after treatment and were used to measure fasting plasma glucose (FPG), HbA_1C, immunoreactive insulin (IRI), and PI. The levels of FPG, HbA_1C, and IRI showed a significant decrease after treatment with Pio in groups P and PG. Treatment with Pio also caused PI to decrease significantly (group P: from 24.7±12.9 (mean±SD) to 14.0±6.2 pmol/L, p<0.01, group PG: from 24.3±11.3 to 14.4±6.5 pmol/L, p<0.01). In group G, treatment with Gli caused FPG and HbA_1C to decrease significantly, but PI showed no change (21.5±12.3 to 21.6±10.4 pmol/L, p=n.s.). In patients with type 2 diabetes, treatment with Pio achieved an improvement of glycemic control and reduced the load on the pancreatic beta cells.

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Patients with Graves' disease (n=61) treated wth propylthiouracil (PTU) or thiamazole (MMI) were studied retrospectively to investigate differences in the prevalence of anti-myeloperoxidase anti-neutrophil cytoplasmic antibodies (MPO-ANCA) in relation to treatment with anti-thyroid drugs. The patients were divided into two groups: PTU-treated group (n=32) and MMI-treated group (n=29). There were no significant differences between the two groups in terms of age, gender distribution, or duration of treatment. In the PTU group, 8/32 (25%) patients were positive for MPO-ANCA, whereas in the MMI group, 1/29 (3.4%) patients were positive. There were no significant differences in age, duration, or dosage between the MPO-ANCA positive and negative patients. Most of the MPO-ANCA positive patients were asymptomatic, except for two patients in whom rheumatic arthritis or membranous glomerulonephritis developed. None of the MPO-ANCA positive patients were diagnosed as having classical ANCA-associated vasculitis. Thus, there is a high frequency of MPO-ANCA in patients with Graves' disease treated with PTU, compared with patients treated with MMI, although classical ANCA-associated vasculitis develops in only a few MPO-ANCA positive patients.

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Vascular leiomyomas are believed to arise from the smooth muscle of blood vessels and are characterized by the proliferation of vascular smooth muscle cells (VSMC) and numerous slit-like vascular lumens. Parathyroid hormone (PTH)-related peptide (PTHrP) plays an important role in local autocrine and/or paracrine regulation of cellular growth and function in VSMC. To investigate the interaction between VSMC and endothelial cells, we evaluated the distribution of PTHrP and PTH/PTHrP-receptor in 10 vascular leiomyomas of the skin by immunohistochemistry and in situ hybridization (ISH) of paraffin-embedded specimens. Both immunohistochemistry and ISH revealed that PTH/PTHrP-receptors are expressed in endothelial cells lining areas with slit-like vascular lumens and very weakly expressed in proliferating VSMC in all vascular leiomyomas. On the other hand, PTHrP itself was localized mainly in proliferating VSMC. These results support the hypothesis that PTHrP acts through the PTH/PTHrP-receptor via an autocrine and/or paracrine mechanism from VSMC to endothelial cells in the formation of characteristic microenvironments of vascular leiomyoma cell composition.

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The Fas/Fas ligand system induces apoptosis, while soluble Fas (sFas) blocks the system and soluble Fas ligand (sFasL) functions to induce apoptosis. The assay of nuclear matrix protein (NMP) released from dead or dying cells can be used to quantitate cell death. Therefore, we evaluated the relationship among serum levels of NMP, sFas, and sFasL in patients with Graves' disease. We measured serum levels of sFas, sFasL, NMP, thyroid hormones and TSH receptor antibody in 20 normal control subjects (5 men, 15 women; mean age, 44.3 years), 32 patients with untreated Graves' disease (4 men, 28 women; mean age, 44.1 years), and 10 patients with Graves' disease treated by methimazole (3 men, 7 women; mean age 39.2 years). Serum NMP was significantly lower (10.4±4.3 IU/ml, p<0.02) in patients with untreated Graves' disease than in patients with treated Graves' disease (16.4±7.3 IU/ml) and control subjects (15.3±8.9 IU/ml). Serum sFas and sFasL were significantly higher in patients with untreated Graves' disease than in patients with treated Graves' disease and in control subjects. In the patient groups with Graves' disease, serum NMP was negatively correlated with sFas (r=-0.612, p<0.001) and serum sFas was positively correlated with FT₄ (r=0.360, p<0.05) and TRAb (r=0.384, p<0.05). Serum NMP was correlated with sFas. These results suggest that serum NMP is decreased in patients with untreated Graves' disease, and that cell death or apoptosis in patients with Graves' disease is affected by soluble Fas under the influence of thyroid function.

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Insulin-like growth factor-I activates a calcium-permeable cation channel GRC (growth factor-regulated channel). In the present study, we investigated the immunohistochemical localization of GRC in human tissues using a polyclonal anti-GRC antibody. Immunoreactive GRC was detected in the stomach, duodenum, large intestine and prostate. In these tissues, GRC-expressing cells were distributed solitarily in the epithelium and coexpressed chromogranin-A, a marker of neuroendocrine cells. GRC was also expressed in the epithelium of the pancreatic duct, mammary gland, parotid gland, and submandibular gland. Epithelial cells of the renal tubule and the tracheal gland were also stained with anti-GRC antibody. In the lung, alveolar macrophages expressed GRC. In the brain, Purkinje cells of the cerebellum and arachnoid cells of the meningis expressed GRC. These results indicate that GRC is expressed in restricted types of cells in particular tissues, and that GRC may modulate calcium entry in these cells.

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The effect of adrenalectomy on the duration of pseudopregnancy was investigated in rats. When adrenalectomy was performed three days before cervical stimulation or when it was done on the first day (day 1, day of estrus) or day 2 of pseudopregnancy, the duration of the pseudopregnancy was significantly prolonged (3.0, 2.5 and 1.8 days respectively). This effect of adrenalectomy was not seen when operation was delayed until day 4 of pseudopregnancy. Adrenalectomy on day 2 of pseudopregnancy significantly increased prolactin (PRL) release at the time of the nocturnal PRL surge (5:00) on day 7. When rats were ovariectomized simultaneously with adrenalectomy on day 1, the stimulating effect of adrenalectomy on PRL release was more evident. The effect of active immunization against corticosterone on the continuation of pseudopregnancy was also examined. Neutralization of plasma corticosterone extended the duration of pseudopregnancy and the binding activity of the antiserum positively correlated with the length of continuing diestrus (P<0.05). These results indicate the negative effect of the adrenal glands, which is probably due to corticosterone, on PRL release in pseudopregnant rats and that the early relief of this inhibition extends the duration of pseudopregnancy.

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Vitamin K is known to mediate carboxylation of glutamyl residues of osteocalcin. We evaluated the effects of vitamin K2 (menatetrenone) treatment (45 mg/day) for 48 weeks on the markers of bone formation and resorption, bone mineral density (BMD), and the incidence of vertebral fractures in 34 Japanese postmenopausal women (aged 48-82 years). Serum levels of alkaline phosphatase (ALP) increased gradually and became significant at 48 weeks after menatetrenone treatment, while urinary excretion of deoxypyridinoline (DPD) decreased transiently but significantly at 4 weeks. Serum levels of both intact osteocalcin (OC) and carboxylated OC (Gla-OC) increased rapidly and significantly within 4 weeks and sustained their high values up to 48 weeks after the treatment, while those of undercarboxylated OC (Glu-OC) decreased reciprocally. These results can be interpreted to suggest that Glu-OC was converted to Gla-OC in vivo. On the other hand, lumbar BMD values showed no significant change and only one subject with a previous vertebral fracture had one newly occurring vertebral fracture. These results indicate that menatetrenone treatment of postmenopausal women constantly elevates bone formation markers as well as converts Glu-OC to Gla-OC. Thus, vitamin K2 treatment may promote bone formation, at least as measured biochemically in these subjects.

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Estrogens play important roles in the development of breast cancer. Inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-1β (IL-1β) exist at high concentrations in breast cancer tissue. Although these cytokines are thought to exert some effect on cancer growth, their precise mechanism is still unclear. In the present study, we investigated the effects of inflammatory cytokines on aromatase (Arom) and steroid sulfatase (STS), which are estrogen-producing enzymes, and cell proliferation using human breast cancer cell lines (SK-BR-3, MCF-7). IL-6 and IL-1β stimulated the activity of Arom and STS. Estrone sulfate (E₁-S) had a stimulus effect on cell proliferation of MCF-7. Although IL-6 did not show significant effect on cell proliferation, cell proliferation was significantly increased when IL-6 and E₁-S were simultaneously added to the incubation medium. This cell proliferative effect was apparently stronger than the addition of E₁-S alone. Addition of IL-1β in the presence of E₁-S also significantly enhanced cell proliferation though IL-1β alone did not show any effect. These results led us to the hypothesis that inflammatory cytokines such as IL-6 and IL-1β regulate proliferation of breast cancer cells through estrogen production by steroid-catalyzing enzymes in the tissue.

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In serum, insulin-like growth factors (IGFs) are primarily present as a ∼150 kDa ternary protein complex, which consists of IGFs, IGF binding protein-3 (IGFBP-3), and acid-labile subunit (ALS). Like IGF-I and IGFBP-3, serum levels of ALS depend on growth hormone (GH). To date, the diagnostic relevance of ALS in adult GH deficiency (GHD) has remained uncertain. To clarify the clinical utility of ALS measurement in adults, we measured serum ALS levels in patients with adult GHD or acromegaly. We also measured the levels of serum IGF-I and IGFBP-3 in these patients to compare the utility of ALS with IGF-I and IGFBP-3 as a marker of GH secretion. Serum ALS was measured by radioimmunoassay (RIA) kit, and serum IGF-I and IGFBP-3 were measured by immunoradiometric assay (IRMA) kits in 56 patients with adult GHD (adult-onset (AO)/child-onset (CO), 13/43) and 43 patients with acromegaly. Serum ALS levels were less than 5^th percentile in 40 of 56 (71%) patients with adult GHD (32/43 (74%) for CO and 8/13 (62%) for AO), and more than 95^th percentile in 38 of 43 (88%) patients with acromegaly, respectively. Serum IGF-I levels were less than -1.96 SD in 43 of 56 (77%) patients with adult GHD (35/43 (81%) for CO and 8/13 (62%) for AO) and more than +1.96 SD in 42 of 43 (98%) patients with acromegaly, respectively. Serum IGFBP-3 levels were less than -1.96 SD in 51 of 56 (91%) patients with adult GHD (42/43 (98%) for CO and 9/13 (69%) for AO) and more than +1.96 SD in 31 of 43 (72%) patients with acromegaly, respectively. These data suggested that measurement of ALS offers no advantage over measurements of serum IGF-I and IGFBP-3. Furthermore, our results indicate that serum IGFBP-3 is the most suitable marker of GH secretion for adult GHD, especially CO, while IGF-I may be the most useful in acromegaly.

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